In a continuing program to study protein conformation and function by semisynthesis, investigations were made into the application of X-ray crystallographic techniques and some other methods to characterize semisynthetic ribonuclease-S' sequence variants. Several deletion and substitution analogues already prepared in this laboratory were successfully crystallized, including the species (des 16-20), (gly 6, des 16020), (p-F-Phe 8, des 16-20), and 4-F-His 12, des 16-20). Microscopic examination and preliminary X-ray diffraction studies indicate conformational similarity between these variably perturbed species, and fully native complex, in the crystalline state. Other aspects of study of 4-F-His 12-containing ribonuclease include proton nuclear magnetic resonance spectroscopic analyses (showing substantially more pH-induced conformational mobility in (4-F-His 12, des 16-20) variant than in native protein); and purification of S-protein and (4-F-His 12, des 16-20) semisynthetic ribonuclease-S' by ribonuclease-specific affinity chromatography, reaffirming the view of active participation of His 12 in catalysis. Affinity chromatography also is being used to determine peptide-protein association of the 4-F-His 12 analogue.